38 research outputs found
Direct Intracellular Delivery of Cell Impermeable Probes of Protein Glycosylation Using Nanostraws
Bioorthogonal chemistry is an effective tool for elucidating metabolic pathways and measuring cellular activity, yet its use is currently limited by the difficulty of getting probes past the cell membrane and into the cytoplasm, especially if more complex probes are desired. Here we present a simple and minimally perturbative technique to deliver functional probes of glycosylation into cells by using a nanostructured “nanostraw” delivery system. Nanostraws provide direct intracellular access to cells through fluid conduits that remain small enough to minimize cell perturbation. First, we demonstrate that our platform can deliver an unmodified azidosugar, N-azidoacetylmannosamine, into cells with similar effectiveness to a chemical modification strategy (peracetylation). We then show that the nanostraw platform enables direct delivery of an azidosugar modified with a charged uridine diphosphate group (UDP) that prevents intracellular penetration, thereby bypassing multiple enzymatic processing steps. By effectively removing the requirement for cell permeability from the probe, the nanostraws expand the toolbox of bioorthogonal probes that can be used to study biological processes on a single, easy-to-use platform
Direct Intracellular Delivery of Cell Impermeable Probes of Protein Glycosylation Using Nanostraws
Bioorthogonal chemistry is an effective tool for elucidating metabolic pathways and measuring cellular activity, yet its use is currently limited by the difficulty of getting probes past the cell membrane and into the cytoplasm, especially if more complex probes are desired. Here we present a simple and minimally perturbative technique to deliver functional probes of glycosylation into cells by using a nanostructured “nanostraw” delivery system. Nanostraws provide direct intracellular access to cells through fluid conduits that remain small enough to minimize cell perturbation. First, we demonstrate that our platform can deliver an unmodified azidosugar, N-azidoacetylmannosamine, into cells with similar effectiveness to a chemical modification strategy (peracetylation). We then show that the nanostraw platform enables direct delivery of an azidosugar modified with a charged uridine diphosphate group (UDP) that prevents intracellular penetration, thereby bypassing multiple enzymatic processing steps. By effectively removing the requirement for cell permeability from the probe, the nanostraws expand the toolbox of bioorthogonal probes that can be used to study biological processes on a single, easy-to-use platform
IL-1R and MyD88 Contribute to the Absence of a Bacterial Microbiome on the Healthy Murine Cornea
Microbial communities are important for the health of mucosal tissues. Traditional culture and gene sequencing have demonstrated bacterial populations on the conjunctiva. However, it remains unclear if the cornea, a transparent tissue critical for vision, also hosts a microbiome. Corneas of wild-type, IL-1R (-/-) and MyD88 (-/-) C57BL/6 mice were imaged after labeling with alkyne-functionalized D-alanine (alkDala), a probe that only incorporates into the peptidoglycan of metabolically active bacteria. Fluorescence in situ hybridization (FISH) was also used to detect viable bacteria. AlkDala labeling was rarely observed on healthy corneas. In contrast, adjacent conjunctivae harbored filamentous alkDala-positive forms, that also labeled with DMN-Tre, a Corynebacterineae-specific probe. FISH confirmed the absence of viable bacteria on healthy corneas, which also cleared deliberately inoculated bacteria within 24 h. Differing from wild-type, both IL-1R (-/-) and MyD88 (-/-) corneas harbored numerous alkDala-labeled bacteria, a result abrogated by topical antibiotics. IL-1R (-/-) corneas were impermeable to fluorescein suggesting that bacterial colonization did not reflect decreased epithelial integrity. Thus, in contrast to the conjunctiva and other mucosal surfaces, healthy murine corneas host very few viable bacteria, and this constitutive state requires the IL-1R and MyD88. While this study cannot exclude the presence of fungi, viruses, or non-viable or dormant bacteria, the data suggest that healthy murine corneas do not host a resident viable bacterial community, or microbiome, the absence of which could have important implications for understanding the homeostasis of this tissue
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Bioorthogonal Chemical Tools Toward the Study of Bacterial Pathogens
In this dissertation, I describe the development of bioorthogonal chemical tools toward the study of bacterial pathogens during infection. Bioorthogonal chemistry has enabled the visualization of biomolecules not amenable to fluorescent protein fusion. By feeding an organism an unnatural metabolite bearing a bioorthogonal chemical reporter, the reporter is incorporated into the cellular biopolymer of interest and can be detected by bioorthogonal reaction with an imaging agent. To adapt this approach towards visualizing pathogenic bacteria in an infection setting, my dissertation research focused on two main areas. First, I developed a family of fluorogenic azide probes activated by bioorthogonal click reactions with alkynes. These probes offer significant advantages in imaging studies where it is challenging to wash away unreacted probe, such as within live cells or in live organisms, the settings in which bacterial pathogens reside. Secondly, I developed a family of unnatural D-alanine analogues bearing cyclooctynes, which are incorporated into nascent peptidoglycan in a wide variety of bacterial species and undergo bioorthogonal reactions with azides. In conjunction with the fluorogenic azide probes I developed, these analogues enabled the visualization of cell wall synthesis in an intracellular bacterial pathogen during infection
Recommended from our members
Bioorthogonal Chemical Tools Toward the Study of Bacterial Pathogens
In this dissertation, I describe the development of bioorthogonal chemical tools toward the study of bacterial pathogens during infection. Bioorthogonal chemistry has enabled the visualization of biomolecules not amenable to fluorescent protein fusion. By feeding an organism an unnatural metabolite bearing a bioorthogonal chemical reporter, the reporter is incorporated into the cellular biopolymer of interest and can be detected by bioorthogonal reaction with an imaging agent. To adapt this approach towards visualizing pathogenic bacteria in an infection setting, my dissertation research focused on two main areas. First, I developed a family of fluorogenic azide probes activated by bioorthogonal click reactions with alkynes. These probes offer significant advantages in imaging studies where it is challenging to wash away unreacted probe, such as within live cells or in live organisms, the settings in which bacterial pathogens reside. Secondly, I developed a family of unnatural D-alanine analogues bearing cyclooctynes, which are incorporated into nascent peptidoglycan in a wide variety of bacterial species and undergo bioorthogonal reactions with azides. In conjunction with the fluorogenic azide probes I developed, these analogues enabled the visualization of cell wall synthesis in an intracellular bacterial pathogen during infection
Tailored silyl ether monomers enable backbone-degradable polynorbornene-based linear, bottlebrush and star copolymers through ROMP
Ring-opening metathesis polymerization of norbornene-based (macro)monomers is a powerful approach for the synthesis of macromolecules with diverse compositions and complex architectures. Nevertheless, a fundamental limitation of polymers prepared by this strategy is their lack of facile degradability, limiting their utility in a range of applications. Here we describe a class of readily available bifunctional silyl ether-based cyclic olefins that copolymerize efficiently with norbornene-based (macro)monomers to provide copolymers with backbone degradability under mildly acidic aqueous conditions and degradation rates that can be tuned over several orders of magnitude, depending on the silyl ether substituents. These monomers can be used to manipulate the in vivo biodistribution and clearance rate of polyethylene glycol-based bottlebrush polymers, as well as to synthesize linear, bottlebrush and brush-arm star copolymers with degradable segments. We expect that this work will enable preparation of degradable polymers by ROMP for biomedical applications, responsive self-assembly and improved sustainability. ©2019, The Author(s), under exclusive licence to Springer Nature Limited.National Institutes of Health (grant no. 1R01CA220468-01
Nanostraws: A Nanofabricated Platform for Delivery of Cell-Impermeable, Synthetic Biomolecules
A fluorogenic screening platform enables directed evolution of an alkyne biosynthetic tool
Approach to the Tricyclic Core of the Tigliane–Daphnane Diterpenes. Concerning the Utility of Transannular Aldol Additions
Two transannular (TA) aldol reactions were used to assemble the tricyclic carbon skeleton found in the tigliane and daphnane classes of diterpene natural products
Bacterial Peptidoglycan Stapling with Functionalized D-Amino Acids
Transpeptidation reinforces the structure of cell wall peptidoglycan, an extracellular heteropolymer that protects bacteria from osmotic lysis. The clinical success of transpeptidase-inhibiting β-lactam antibiotics illustrates the essentiality of these cross-linkages for cell wall integrity, but the presence of multiple, seemingly redundant transpeptidases in many bacterial species makes it challenging to determine cross-link function precisely. Here we present a technique to covalently link peptide strands by chemical rather than enzymatic reaction. We employ bio-compatible click chemistry to induce triazole formation between azido- and alkynyl-D-alanine residues that are metabolically installed in the cell walls of Gram-positive and Gram-negative bacteria. Synthetic triazole cross-links can be visualized by substituting azido-D-alanine with azidocoumarin-D-alanine, an amino acid derivative that undergoes fluorescent enhancement upon reaction with terminal alkynes. Cell wall stapling protects the model bacterium Escherichia coli from β-lactam treatment. Chemical control of cell wall structure in live bacteria can provide functional insights that are orthogonal to those obtained by genetics.<br /